Microorganisms in the production of biochemicals

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Many products of microbial metabolism find an application in the food and other industries. These include amino acids, steroids, enzymes and antibiotics (Table 17.4). Microbial growth conditions are adjusted so that production of the metabolite in question takes place at an optimal rate. Often an unnaturally high rate of production is achieved by the use of a mutated or genetically engineered strain of microorganism, or by manipulating culture conditions to favour excess metabolite production.

The development of a microbial means of producing acetone was vital to the allied effort in the First World War. Acetone was a crucial precursor in explosives manufacture and the demands of war soon outstripped supply by traditional methods. The problem was solved when Chaim Weismann isolated a strain of Clostridium acetobutylicum that could ferment molasses to acetone and butanol (another industrially useful product). Nowadays, acetone is made more cheaply from petrochemicals.

Microbially produced amino acids are used in the food industry, in medicine and as raw materials in the chemical industry. The one produced in the greatest quantities by far is glutamic acid (in excess of half a billion tonnes per year), with most of it ending up as the flavour enhancer monosodium glutamate. The amino acids aspartic acid and phenylalanine are components of the artificial sweetener aspartame and are also synthesised on a large scale.

Table 17.4 Some industrial applications of microbially produced enzymes

Industry

Enzyme

Application

Food & drink

Rennin Lipase Pectinase

Amylase

Amylase Glucoamylase Glucose isomerase

Cheese manufacture

Fruit juice production Coffee bean extraction Improved bread dough quality Haze removal in beer Fructose syrup production

Animal feed

Amylase Cellulase Protease

Improved digestibility

Detergent

Protease Lipase Amylase Cellulase

Stain and grease removal Fabric softener

Paper

Cellulase

Pulp production

Textile

Cellulase

'Stone-washed' jeans

Leather

Protease Lipase

Dehairing, softening, fat removal

Molecular biology

Taq polymerase

Polymerase chain reaction

A number of organic acids are produced industrially by microbial means, most notably citric acid, which has a wide range of applications in the food and pharmaceutical industries. This is mostly produced as a secondary metabolite by the large-scale culture of the mould Aspergillus niger.

Certain microorganisms serve as a ready source of vitamins. In many cases these can be synthesised less expensively by chemical means; however, riboflavin (by the mould Ashbya gossypii) and vitamin B12 (by the bacteria Propionibacterium shermanii and Pseudomonas denitrificans) are produced by large-scale microbial fermentation. Microorganisms play a partial role in the production of ascorbic acid (vitamin C). Initially, glucose is reduced chemically to sorbitol, which is then oxidised by a strain of Acetobacter suboxydans to the hexose sorbose. Chemical modifications convert this to ascorbic acid (Figure 17.4).

Enzymes of fungal and bacterial origin have been utilised for many centuries in a variety of processes. It is now possible to isolate and purify the enzymes needed for a specific process and the worldwide market is currently worth around a billion pounds. The most useful industrial enzymes include proteases, amylases, lipases

If you wear contact lenses, you have probably used an enzyme preparation to remove protein deposits.

I Chemical

HO CH

CH2OH HO CH HO CH

HC OH HO CH

CH2OH D-Sorbitol

CH2OH CO HO CH

HC OH HO CH

CH2OH L-Sorbitol

HO CH

L-Ascorbic Acid

Figure 17.4 Ascorbic acid is produced by a combination of chemical and microbial reactions Most steps in the synthesis of ascorbic acid are purely chemical, but the conversion of sorbitol to sorbose is carried out by the sorbitol dehydrogenase enzyme of Acetobacter suboxydans, with NAD+ acting as an electron acceptor

CH2OH

and pectinase (Table17.4). Some applications of enzymes are listed in Table 17.5, and two examples are briefly described below.

Syrups and modified starches are used in a wide range of foodstuffs, including soft drinks, confectionery and ice cream, as well as having a wealth of other applications. Different enzymes or combinations of enzymes are used to produce the desired consistencies and physical properties. High fructose corn syrup (HFCS) is a sweetener used in a multitude of food products. It is some 75 per cent sweeter than sucrose and has several other advantages. HFCS is a mixture of fructose, dextrose (a form of glucose) and disaccharides, and is produced by the action of a series of three enzymes on the starch (amylose and amylopectin) of corn (maize). Alpha amylase hydrolyses the internal a-1, 4-glycosidic bonds of starch, but is not able to degrade ends of the chain. The resulting di- and oligosaccharides are broken down to the monomer glucose by the action of glucoamylase, then finally glucose isomerase converts some of the glucose to its isomer, fructose.

Enzymes have been added to cleaning products such as washing powders, carpet shampoos and stain removers since the 1960s, and this remains one of the principal

Table 17.5 Commercially useful products of microbial metabolism

Product

Amino acids: Glutamic acid Lysine

Aspartic acid & phenylalanine

Citric acid

Enzymes

Antibiotics

Vitamins

Steroids

Flavour enhancer Animal feed additive Artificial sweetener (aspartame) Antioxidant, flavour enhancer, emulsifier Numerous - see Table 17.4 Treatment of infectious diseaases Dietary supplements Anti-inflammatory drugs, oral contraceptives industrial applications of enzymes. Proteases are the most widely used enzymes in this context; working in combination with a surfactant, they hydrolyse protein-based stains such as blood, sweat and various foods. Greasy and oily stains present a different challenge, made all the more difficult by the move towards lower washing temperatures. The inclusion of lipases aids the removal of stains such as butter, salad dressing and lipstick, while amylases deal with starch-based stains such as cereal or custard. The food and detergent industries between them account for around 80 per cent of all enzyme usage.

We have already seen in Chapter 14 that antibiotics are now produced on a huge scale worldwide. Figure 17.5 outlines the stages in the isolation, development and production of an antibiotic.

Isolating an antibiotic from a natural source is not all that difficult, but finding a new one that is therapeutically useful is another matter. Initially, the antimicrobial properties of a new isolate are assessed by streaking it across an agar plate, then inoculating a range

Isolation and purification

Assessment of antimicrobial efficiency

Small-scale production

Toxicity testing

Pilot-scale production

Clinical trials

Figure 17.5 Stages in the isolation and development of an antibiotic. See the text for details

Isolate streaked across agar plate

Several bacterial strains Inhibition of streaked onto plate some strains by antibiotic

Several bacterial strains Inhibition of streaked onto plate some strains by antibiotic

Figure 17.6 Assessing the antimicrobial properties of an antibiotic. Candidate antibiotic is streaked onto an agar plate along with several bacterial isolates. Following incubation, areas of clearing indicate inhibition of growth and thus susceptibility to the antibiotic

of bacteria at right angles (Figure 17.6). As the antibiotic diffuses through the agar, it will inhibit growth of any susceptible species. Isolates that still show potential are then grown up in a laboratory scale fermenter; it is essential for commercial culture that the antibiotic-producing organism can be cultured in this way.

Before committing to large-scale production, exhaustive further tests must be carried out on two fronts: to ascertain the potency of the preparation and the breadth of its antimicrobial spectrum, and to determine its therapeutic index (see Chapter 14) by carrying out toxicity testing on animals. The final stages of development involve pilot-scale production, followed by clinical trials on human volunteers.

When an antibiotic or any other fermentation product finally goes into production, it is cultured in huge stirred fermenters or bioreactors, which may be as large as 200 000 litres. A typical stirred fermenter has impellers for mixing the culture, an air line for aeration and microprocessor-controlled probes for the continuous monitoring and regulation of temperature, pH and oxygen content (Figure 17.7). Cultures with a high protein content may also have an antifoaming agent added. The process of scale-up is a complex operation, and not simply a matter of growing the microorganism in question in ever-larger vessels. Factors such as temperature, pH, aeration, must all be considered at the level of the individual cell if scale-up is to be successful. Fermenters are usually made from stainless steel, which can withstand heat sterilisation; the economic consequences of microbial contamination when working on such a large scale can be immense.

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